A paper copy of the sequence listing and a computer readable form of the same sequence listing are appended below and herein incorporated by reference. The information recorded in computer readable form is identical to the written sequence listing, according to 37 C.F.R. 1.821 (f).
1. Field of the Invention
The invention relates generally to methods of detecting and quantifying specific proteins, in particular sequence-specific DNA binding proteins, by changes in luminescence signal intensity or changes in color due to the processing of a calorimetric substrate. The invention is used in any application where the detection or quantification of DNA binding activity of a DNA binding protein is desired.
2. Description of the Related Art
The ability to detect and quantify specific protein molecules is of great importance in basic research and in clinical applications. Determination of the level of a specific protein is one of the most useful and important experimental procedures in biomedical research and molecular diagnostics. Cellular levels of specific proteins are commonly used as diagnostic markers for many diseases.
Protein-nucleic acid interactions are an extremely important and physiologically relevant type of macromolecular contact found in the cell. Many proteins that play an important role in regulating many cellular processes possess natural sequence-specific DNA binding activity. These proteins include transcription factors, chromatin remodeling factors and DNA maintenance enzymes. For a review of DNA binding proteins, see Benjamin Lewin, Genes VII, Oxford University Press, New York, 2000, which is herein incorporated by reference.
Transcription factors bind to specific cognate DNA elements, which include promoters, enhancers and silencer elements. They may be activators, repressors or both, depending on the cellular context, whose levels are important for regulation of gene expression. Thus, many of these proteins are important in disease development and disease diagnosis. For example, several transcription factors, which when overexpressed or inappropriately expressed, are oncogenes. These oncogenic transcription factors include myc, myb, fos, jun, rel and erb. Another cancer related transcription factor, p53, is involved in development of many cancers (Ko, L. L., and Prives, C. Genes Dev. 10, 1054-1072, 1996).
Chromatin remodeling factors are also important for the regulation of gene expression. Generally, regions of highly condensed chromatin, called heterochromatin, contain genes which are not actively transcribed, whereas regions of loose or non-condensed chromatin, called euchromatin, contain genes that are actively transcribed. During cellular differentiation, cancerous transformation and normal physiological homeostasis, chromatin may be remodeled. That is, some chromosomal regions become inaccessible to transcription factors and RNA polymerase, whereas other regions become accessible. Several DNA binding factors are involved in this dynamic process including nucleosome proteins (e.g., histones), histone acetyltransferases, histone deacetylases, DNA methyltransferases, nucleoplasmins, HMG proteins, repressor complex proteins, polycomb-related factors and trithorax-related factors.
DNA maintenance enzymes are DNA binding proteins necessary for the repair of damaged DNA, faithful replication of DNA and exchange of genetic information during recombination. Several types of cancer and other disease syndromes are the result of defective DNA maintenance enzymes. For example, Xeroderma pigmentosum, a horrific genetic disease whereby the sufferer is predisposed to skin cancer, is due to defective nucleotide-excision repair enzymes. Hereditary non-polyposis colorectal cancer is caused in large part by defective mismatch repair enzymes. Some forms of hereditary breast cancers are due to defective homologous recombination enzymes. For a review of genome maintenance systems and their role in cancer, see Hoeijmakers, J. H. J., Nature 411, 366-374, 2001, which is herein incorporated by reference. Thus, there is a significant interest in convenient and accurate methods for detecting, monitoring and/or quantifying DNA binding activity of DNA binding proteins.
The most common approaches taken to detect proteins exhibiting sequence-specific DNA binding activity are gel shift assays and various DNA footprinting assays (Fried, M.G., and Crothers, D. M. Nucleic Acids Res. 9, 6505-6525, 1981; Galas, D. J., and Schmitz, A. Nucleic Acid Res. 5, 3157-3170, 1978). These methods are laborious and time-consuming procedures, which typically involve the use of dangerous and expensive radioisotopes. Furthermore, these methods are not generally adaptable to high-throughput assay formats. Different fluorescence based methodologies for detecting and studying DNA binding proteins have been developed to overcome the deficiencies of gel shift and DNA footprinting assays.
Detection of molecules by fluorescence has several important advantages compared to alternative detection methods. Fluorescence provides an unmatched sensitivity of detection, as demonstrated by the detection of single molecules using fluorescence (Weiss, S. Science 283, 1676-1683, 1999). Detection of fluorescence, changes in fluorescence intensity or changes in emission spectra can be easily achieved by the selection of specific wavelengths of excitation and emission. Fluorescence provides a real-time signal allowing real-time monitoring of processes and real-time cellular imaging by microscopy (see Lakowicz, J. R. Principles of Fluorescence Spectroscopy, Kluwer Academic/Plenum Press, New York, 1999, which is herein incorporated by reference). Additionally, well-established methods and instrumentation for high-throughput detection of fluorescence signals exist in the art.
Current methods for detecting DNA binding proteins in solution using fluorescence rely on one of the following phenomena: (i) a change in the fluorescence intensity of a fluorochrome (also called a fluorophore or a fluorescent probe or label), which is present either on the protein or on the DNA, as a result of the perturbation of the microenvironment of the probe upon protein-DNA complex formation; (ii) a change of fluorescence polarization of the fluorochrome, which is present either on the protein or on the DNA, as a result of an increase in the molecular size of the protein-DNA complex relative to the unbound DNA or protein molecules; and (iii) resonance energy transfer between one fluorochrome present in DNA and another fluorochrome present in a protein as a result the proximity between DNA and the protein in protein-DNA complex. For a review on methods of detecting fluorescence signal detection, see Hill, J. J., and Royer, C. A. Methods in Enzymol. 278, 390-416, 1997, which is herein incorporated by reference.
In the first group of methods (group i), the change in the fluorescence signal is the result of a change in the microenvironment of the fluorescence probe which occurs upon the formation of a protein-DNA complex. Since the generation of the change in the fluorescence signal relies on the unpredictable chance that the formation of a protein-DNA complex will in fact change the environment of the fluorescence probe significantly enough to provide a measurable change in fluorescence, this approach is not generally applicable in that it will work in some cases but not in others. The outcome of such an assay depends on the nature of the protein, DNA sequence, the length of DNA fragment, identity of the fluorescence probe used, and the method of attachment of the fluorescence probe to DNA. Therefore, it is essentially impossible to predict when this method will or will not work since the mechanisms of the changes of fluorescence intensity due to the change in probe environment are not well understood. Examples of the application of this idea to the detection of protein-DNA complexes using fluorochromes attached to the protein or the DNA can be found in the following technical literature, which are herein incorporated by reference (Sha, M., Ferre-D""Amare, Burley, S. K., and Goss, D. J. J. Biol. Chem. 270, 19325-19329, 1995; Reedstrom, R. J., Brown, M. P., Grillo, A., Roen, D, and Royer, C. A. J. Mol. Biol. 273, 572-585, 1997; Erickson, G. H, and Daksis, J. WO 00/40753).
The unpredictability of such an assay format is illustrated in the literature. Some published studies demonstrate a significant change of fluorescence intensity upon protein-DNA complex formation. For example, a 50% quenching of fluorescein-labeled DNA was observed upon binding to the Trp repressor protein, and a similar degree of quenching was also observed upon glucocorticoid receptor binding to several different DNA targets (Reedstrom, R. J., Brown, M. P., Grillo, A., Roen, D, and Royer, C. A. J. Mol. Biol. 273, 572-20 585, 1997; Hill, J. J., and Royer, C. A. Methods in Enzymol. 278, 390-416, 1997). In other reports, either only small quenching or small increases of fluorescence emission have been observed (Bjornson, K. P., Moore, K. J. M., and Lohman, T. M. Biochemistry 35, 2268-2282, 1996; Hey, T., Lipps, G., and Krauss, G. Biochemistry 40, 2901-2910, 2001; Bailey, M., Hagmar, P., Millar, D. P., Davidson, B. E., Tong, G., Haralambidis, J., and Sawyer, W. H. Biochemistry 34, 15802-15812, 1995; Parkhurst, K. M., Brenowitz, M., and Parkhurst, L. J. Biochemistry 35, 7459-7465, 1996; Wang, K., Rodgers, M. E., Toptygin, D., Munsen, V. A., and Brand, L. Biochemistry 37, 41-50, 1998). Finally, in many reports no change of fluorescence intensity upon binding of the protein to the fluorochrome-labeled cognate nucleic acid was observed (Bailey, M., Hagmar, P., Millar, D. P., Davidson, B. E., Tong, G., Haralambidis, J., and Sawyer, W. H. Biochemistry 34, 15802-15812, 1995; Gourves, A. S., LeGac, N. T., Villani, G., Boehmer, P. E., and Johnson, N. P. J. Biol. Chem. 275, 10864-10869, 2000; Hey, T., Lipps, G., and Krauss, G. Biochemistry 40, 2901-2910, 2001; Lima, L. M. T. R., Foguel, D., and Silva, J. L. Proc. Natl. Acad. Sci USA, 97, 14289-14294, 2000; Ozers, M. S., Hill, J. J., Wood, E. K., Nardulli, A. M., Royer, C. A., and Gorski, J J. Biol. Chem. 272, 30405-30411, 1997; Reedstrom, R. J., Brown, M. P.,Grillo, A., Roen, D, and Royer, C. A. J. Mol. Biol. 273, 572-585, 1997; Wang, K., Rodgers, M. E.,Toptygin, D., Munsen, V. A., and Brand, L. Biochemistry 37, 41-50, 1998).
The lack of the predictability of the outcome of this assay format is perhaps best illustrated by the work described by Bailey et al. (supra), which examines the change in fluorescence of a DNA molecule labeled with fluorescein at eight different positions in response to binding of the TyrR protein. A change of fluorescence intensity was observed with only one specific DNA construct, whereas in the seven remaining cases no change of fluorescence intensity was observed.
Another weakness of the change-in-fluorescence-intensity format is that the range of changes of the fluorescence signal is very limited. In the most favorable cases, the observed quenching was 60-70%, whereas in the majority of the cases reported the observed quenching (or enhancement) was less than or equal to 30%. While 60-70% quenching is sufficient for a practical assay, less than or equal to 30% quenching is not large enough for practical applications. Furthermore, fluorescence-quenching assays are limited in the selection of useful fluorescence probes. In many applications it is advantageous to be able to use a variety of fluorescent colors, which allows for the use of signal enhancement or the ratio between signals at different wavelengths.
Another type of fluorescence-based detection assay, called fluorescence polarization, has also been extensively used for the detection of protein-DNA complex formation (see Heyduk, T., and Lee, J. C. Proc. Natl. Acad. Sci USA 87, 1744-1748, 1990, which is herein incorporated by reference). The physical basis of this approach is that the fluorescence polarization signal of a macromolecule labeled with a fluorochrome depends on the size of the macromolecule (Lakowicz, J. R. Principles of Fluorescence Spectroscopy, Kluwer Academic/Plenum Press, New York, 1999, herein incorporated by reference). Hence, upon the formation of a protein-DNA complex from the protein and DNA components, a larger molecular entity is created, which has an altered fluorescence signature. The use of fluorescence polarization to detect protein-DNA complexes is described in Royer (1998, U.S. Pat. No. 5,756,292), which is herein incorporated by reference. The limitations of the fluorescence polarization approach include the small dynamic range of fluorescence polarization change, the applicability to only relatively short DNA molecules, and the susceptibility to artifacts due to light scattering. Furthermore, fluorescence polarization requires the use of specialized instrumentation and, as in the method described above, the outcome of the fluorescence polarization experiment is sometimes difficult to predict. For example, Hill and Royer (Methods in Enzymol. 278, 390-416, 1997, which is herein incorporated by reference) describe an experiment in which no change in fluorescence polarization signal was detected even though the formation of the protein DNA complex had been shown by other techniques.
A third fluorescence-based assay for the detection of the protein-DNA complex formation is resonance energy transfer (FRET) (Stryer, L.Ann. Rev. Biochem. 47, 819-846, 1978, which is herein incorporated by reference). FRET is based upon the transfer of emitted light energy from a fluorochrome (fluorescent donor) to an acceptor molecule (fluorescent acceptor), which may also be a fluorochrome. The FRET assay is based on the difference in the proximity between DNA labeled with one fluorochrome and the protein labeled with another fluorochrome, wherein the physical proximity between the two fluorochromes in the protein-DNA complex is greater than between the free protein and free DNA. Several published reports illustrate the use of this approach to detect and study protein-DNA interactions (see Kane, S. A., Fleener, C. A., Zhang, Y. S., Davis, L. J., Musselman, A. L., and Huang, P. S. Anal. Biochem. 278, 29-39, 2000, which is herein incorporated by reference). The major limitation of the FRET approach is that both the DNA and the protein need to be modified with fluorescence probes.
In summary, luminescence or fluorescence-based assay systems are an attractive tool for detecting DNA binding proteins. However, a general, inexpensive, simple, multicolor fluorescence or luminescence method for detecting sequence specific DNA binding proteins which would be compatible with high-throughput detection formats is currently not available.
Disclosed are methods of detecting and quantifying DNA binding proteins based upon proximity-based luminescence transfer. In one embodiment of the invention, two double-stranded oligonucleotides are synthesized or isolated, such that, by combining the two double-stranded oligonucleotides, a complete DNA element is formed across the juncture of the oligonucleotides (see FIG. 1A). The DNA binding element comprises a cognate sequence for the binding of DNA binding factors. The first oligonucleotide is labeled with a fluorophore, which is hereafter referred to as the xe2x80x9cfluorescent donorxe2x80x9d, and the second oligonucleotide is labeled with a fluorescent quenching molecule, which is hereafter referred to as xe2x80x9cfluorescent acceptorxe2x80x9d, wherein said quenching molecule may be another fluorophore of a lower excitation wavelength than the first fluorophore. The fluorescent-labeled oligonucleotides are mixed with a sample, which contains a DNA binding factor. Upon mixing, the DNA binding factor associates with both portions of its cognate DNA element, thereby stabilizing the association of the two oligonucleotides. When the two oligonucleotides are in close proximity, the fluorescent donor of the first oligonucleotide transfers its emitted light energy to the fluorescent acceptor of the second oligonucleotide, resulting in the quenching of the emitted light from the fluorescent donor. Fluorescence is measured using standard spectrophotometric or fluorometric methods that are well known in the art. The quenching of the fluorescent signal is correlated with the association of the DNA binding factor to the cognate DNA element.
Given that fluorescence and fluorescence quenching can be routinely measured with accuracy and precision, the present invention is used to quantify the amount or specific activity of a DNA binding factor in a sample, quantify the dissociation constant or affinity of a DNA binding factor, as well as detect the presence of a DNA binding factor in a sample by measuring the change in fluorescence wavelength or intensity.
In one embodiment, the labeled oligonucleotides that comprise a DNA binding element (also known as nucleic acid components) are in solution and free to diffuse in all directions. In another embodiment, said oligonucleotides are affixed to a solid phase substrate, such as, for example, a microtiter plate, microarray slide, membrane or microsphere. In another embodiment, each pair or set of matched oligonucleotides are connected via a linker molecule, wherein the first oligonucleotide is linked to the second oligonucleotide by way of a linker molecule attached to the end of each oligonucleotide, which end is distal to the DNA binding element or fluorescently tagged end of each oligonucleotide. The linked oligonucleotide pairs may be affixed to a solid phase substrate, such as a microtiter plate, membrane, microarray device or microsphere, or they may be free to diffuse in solution.
In another embodiment, a single polynucleotide is labeled in two positions, with a fluorescent donor at the first position and with a fluorescent acceptor at the second position, wherein the fluorescent labels are at such a distance from one another so as not to interact spectroscopically in the absence of a bridging DNA binding factor. In one aspect of this embodiment a portion of a DNA element is located near the first position and another portion of the same DNA element is located near the second position. Upon the binding of a DNA binding factor to both portions of said element, the first position is brought into proximity to the second position, thereby facilitating or stabilizing the spectroscopic interaction between fluorescent donor and fluorescent acceptor. In another aspect of this embodiment, a first DNA element is located at or near the first position and a second DNA element is located at or near the second position. Upon the binding of a DNA binding factor or complex assembly of DNA binding factors (as in an enhanceosome, for example) to the first and/or second element, the first position is brought into proximity to the second position, thereby facilitating or stabilizing the spectroscopic interaction between fluorescent donor and fluorescent acceptor, resulting in a measurable change in fluorescence due to fluorescent energy transfer or quenching.
Any method of proximity-based luminescence detection can be used in the present invention. Embodiments of proximity-based or coincident-based luminescence detection methods include, but are not limited to fluorescence energy transfer, luminescence resonance energy transfer, fluorescence cross-correlation spectroscopy, flow cytometry, direct quenching, ground-state complex formation, chemiluminescence energy transfer, bioluminescence energy transfer and excimer formation. It is understood that the skilled artisan would recognize alternative proximity-based luminescence detection methods that are applicable to the present invention and are herein included in this invention.
Any fluorophore may be used as a fluorescent donor or acceptor in the present invention, however it is preferred that the acceptor excitation wavelength matches the emission wavelength of the donor. In another embodiment, a quencher molecule may be used as a fluorescence acceptor, wherein no light is emitted from the quencher upon excitation. Examples of fluorophores and quenchers are included in the group consisting of Alexa Fluor(copyright) 350, Alexa Fluor(copyright) 430, Alexa Fluor(copyright) 488, Alexa Fluor(copyright) 532, Alexa Fluor(copyright) 546, Alexa Fluor(copyright) 568, Alexa Fluor(copyright) 594, Alexa Fluor(copyright) 633, Alexa Fluor(copyright) 647, Alexa Fluor(copyright) 660, Alexa Fluor(copyright) 680, 7-diethylaminocoumarin-3-carboxylic acid, Fluorescein, Oregon Green 488, Oregon Green 514, Tetramethylrhodamine, Rhodamine X, Texas Red dye, QSY 7, QSY33, Dabcyl, BODIPY FL, BODIPY 630/650, BODIPY 650/665, BODIPY TMR-X, BODIPY TR-X, Dialkylaminocoumarin, Cy5.5, Cy5, Cy3.5, Cy3, DTPA(Eu3+)-AMCA and TTHA(Eu3+)-AMCA. It is understood that the skilled artisan would recognize that any compatible fluorescence donor/acceptor pair will work in the present invention and that the aforementioned fluorophores and quenchers are exemplary and not limiting.
In another embodiment, it is envisioned that, in addition to luminescence-based proximity assays, flow cytometry, or colorimetric enzyme-based assays may be used to detect binding of a DNA binding factor to a cognate DNA element. In fluorescence assisted cell sorting, one nucleic acid component is coupled to a bead or microsphere and the other nucleic acid component is coupled to a luminescent molecule or fluorochrome.
In another embodiment, the present invention is used to diagnose and or characterize disease states by profiling the activity of various diagnostic DNA binding proteins in a sample obtained from a patient. It is envisioned that some diseases involve the misexpression of DNA binding factors. For example, some cancers involve the overexpression of transcription factors such as c-myc, c-fos, c-jun, rel or erbA (see Genes IV by Lewin, p. 890), while other cancers, for example some types of breast cancer or colorectal cancers, underexpress DNA repair enzymes. In this embodiment, biopsy samples are combined with labeled oligonucleotides or nucleic acid components, as herein described above, to assay for the presence, absence or specific activity of specific DNA binding factors.
In another embodiment, the present invention is directed to a method of detecting and/or quantifying cell regulatory factors in a sample, wherein said cell regulatory factors act as cofactors or coenzymes that facilitate or abrogate the association of DNA binding factors to cognate DNA elements. A test sample that may contain a regulatory factor is combined with a mixture or kit comprising the labeled oligonucleotides or polynucleotides of the present invention (supra) and the cognate DNA binding factor, wherein the DNA binding activity of the DNA binding factor depends fully or in part on the presence or absence of said regulatory factor. It is envisioned that if the DNA binding factor requires the presence of said regulatory factor in order to bind to the cognate DNA element, fluorescence energy transfer or quenching will occur when the regulatory factor is present in the sample. It is likewise envisioned that if said regulatory factor interferes with the binding of the DNA binding factor to its cognate DNA element, fluorescence energy transfer or quenching will not occur.
In another embodiment, the present invention is drawn to a method of identifying agents or drugs that affect the binding of DNA binding factors to DNA elements. In a situation analogous to the method of detecting and/or quantifying cell regulatory factors in a sample (supra), prospective agents or drugs are combined with various sets of DNA binding factors and labeled oligonucleotides or nucleic acid components comprising cognate DNA elements. In the event that the agent or drug inhibits or disrupts interaction of the DNA binding factor with the DNA element, no change in fluorescence would be measured. In the event that the agent or drug augments the binding of the DNA binding factor to the DNA element, an enhancement of the fluorescence energy transfer or change in fluorescence would be measured.
In another embodiment, the invention is drawn to an array device comprising multiple pairs of labeled oligonucleotides affixed to a solid matrix or suspended in solution in a linear or multidimensional format. Cognate pairs of labeled oligonucleotides, wherein each oligonucleotide comprises a portion of a DNA element that is a binding site for a DNA binding factor and the first label is a fluorescent donor molecule or a chemiluminescent or calorimetric substrate and the second label is a fluorescent acceptor or catalyst for the chemiluminescent or calorimetric substrate, are affixed to a specific position on a solid substrate or suspended within a specific well of a multi-well plate. The solid substrate may be a membrane, such as, for example nitrocellulose, nylon or polyvinyidifluoride (xe2x80x9cPVDFxe2x80x9d), a multi-well plate or another convenient substrate that lends itself to this purpose. In another aspect of this embodiment, each cognate oligonucleotide pair is linked together by way of a linker molecule affixed to the end of each oligonucleotide distal to the label and DNA element or portion thereof. The linked oligonucleotide pairs are affixed to the solid matrix in a specific array format or are placed within specific wells of a multi-well plate. In another aspect of this embodiment, the array device comprises several nucleic acid components displayed in an array format, wherein each polynucleotide comprises one or several DNA elements that are labeled, wherein the first label is a fluorescent donor molecule or a chemiluminescent or colorimetric substrate and the second label is a fluorescent acceptor or catalyst for the chemiluminescent or calorimetric substrate. Each specific polynucleotide is affixed to a specific position on a solid substrate or suspended within a specific well of a multi-well plate, as described for the oligonucleotide pairs (supra).
The above summary describes in brief the preferred embodiments of the present invention and is not intended to limit the scope of the invention to these described embodiments. The skilled artisan will recognize that there are other possible embodiments of this invention which utilize the general principle of proximity chemical reactions to identify agents that are involved in DNA binding.